Zoopharmacognosy in Diseased Laboratory Mice: Conflicting Evidence

Minesh Kapadia1, Hui Zhao1, Donglai Ma2, Rupal Hatkar1, Monica Marchese3, Boris Sakic1* 1 Department of Psychiatry and Behavioural Neurosciences, McMaster University, Hamilton, Ontario, Canada, 2 Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada, 3 Department of Clinical Epidemiology and Biostatistics, McMaster University, Hamilton, Ontario, Canada

Abstract Zoopharmacognosy denotes a constellation of learned ingestive responses that promote healing and survival of infected or poisoned . A similar self-medication phenomenon was reported in diseased laboratory rodents. In particular, a series of studies revealed that autoimmune MRL/lpr mice readily consume solutions paired or laced with (CY), an immunosuppressive drug that prevents inflammatory damage to internal organs. However, due to design limitations, it could not be elucidated whether such a response reflects the learned therapeutic effect of CY, or a deficit in sensory input. We presently assess the behavioural effects of prolonged consumption of CY-laced, 16% sucrose solution in a continuous choice paradigm, with tap water available ad lib. Contrary to overall expectation, MRL/lpr mice did not increase their intake of CY with disease progression. Moreover, they ingested lower doses of CY and preferred less CY-laced sucrose solution than age-matched controls. The results obtained could not confirm zoopharmacognosy in diseased MRL/lpr mice, likely due to impaired responsiveness to palatable stimulation, or attenuated survival mechanisms after prolonged inbreeding in captivity. However, by revealing the effectiveness of unrestricted drinking of drug-laced sucrose solution on behavior and immunity, the current study supports broader use of such an administration route in behavioural studies sensitive to external stressors.

Citation: Kapadia M, Zhao H, Ma D, Hatkar R, Marchese M, et al. (2014) Zoopharmacognosy in Diseased Laboratory Mice: Conflicting Evidence. PLoS ONE 9(6): e100684. doi:10.1371/journal.pone.0100684 Editor: Judith Homberg, Radboud University, Netherlands Received March 19, 2014; Accepted May 27, 2014; Published June 23, 2014 Copyright: ß 2014 Kapadia et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. Funding: This project was funded by PhD scholarship from the Father Sean O’Sullivan Foundation (St. Joseph’s hospital, Hamilton, Ontario) to Minesh Kapadia. The Father Sean O’Sullivan Foundation had no role in the in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * Email: [email protected]

Introduction Age-matched congenic MRL/MpJ +/+ (MRL +/+) controls develop a mild form of the disease and have a life span of up to two The term zoopharmacognosy is derived from Greek words zoo years [15]. Three studies by Grota and colleagues suggested that (), pharma (drug), and gnosy (knowing), denoting an evolu- MRL/lpr mice adopt a pattern similar to wild animals to tionary phenomenon comprised of mostly ingestive behaviors restore homeostasis within the immune system. Initial evidence aimed at preventing or reducing the harmful effects of pathogens came from a conditioning study, which utilized repeated pairings and toxins. Particularly, when sick, certain wild animals will ingest of access to a palatable stimulus (undiluted chocolate milk) with herbs, , dirt, or that have medicinal properties [1]. injections of a noxious, immunosuppressive drug cyclophospha- Great apes often consume with non-nutritional values that mide (CY) after 23-hr fluid deprivation. Compared to MRL +/+ have beneficial effects on gut acidity [2,3], or combat intestinal controls which manifest a mild form of disease, severely sick parasitic infection [4–6]. Similarly, domestic horses, dogs, cats, MRL/lpr mice did not show strong taste aversion learning when and cows infected with a pathogen may ingest herbs containing exposed to the 1-hr ‘‘chocolate milk vs. tap water’’ preference test nutrients and chemicals with anti-parasitic properties [7]. More [16]. This ‘‘learning deficit’’ was not observed prior to the interestingly, cattle and some birds eat -rich termite mound development of systemic disease, or when non-immunosuppressive soil, which deactivates ingested pathogens or fruit toxins [8,9]. unconditioned stimuli were used, suggesting MRL/lpr mice rather Although the underlying psychological and physiological mecha- associated chocolate milk with relief of autoimmune symptoms nisms of such learned self-medicating behaviour are unclear, its adaptive value is proposed to be widespread [10], encompassing following pairings with CY. In an extension of their work, the laboratory animals. same group demonstrated that fluid-deprived MRL/lpr mice The MRL/MpJ-Faslpr/2J (MRL/lpr) murine substrain sponta- ‘‘voluntarily’’ consumed more CY dissolved in chocolate milk than neously develops systemic autoimmune disease with clinical and age-matched congenic MRL +/+ controls [17]. Consistent with serological manifestations reminiscent of human systemic lupus the results from the conditioning study, consumption of CY erythematosus, SLE [11]. Due to a lpr mutation on chromosome solution differed between the two MRL substrains only when 19 and dysfunctional Fas receptor in negative selection of severe autoimmune symptoms in MRL/lpr mice emerged. autoreactive T cells [12,13], MRL/lpr mice develop florid disease Considering that dilutions of chocolate milk and fluid deprivation by 3 months, with few surviving beyond 6 months of age [14]. did not have a significant effect on CY consumption, it was

PLOS ONE | www.plosone.org 1 June 2014 | Volume 9 | Issue 6 | e100684 Zoopharmacognosy in Laboratory Animals proposed that autoimmune mice learned to consume more CY in by the local Animal Care Committee (McMaster University AUP an effort to correct their immune system dysregulation [18]. #11-03-11) and performed in accordance to guidelines set out by Although some classical conditioning studies have shown that the Canadian Council of Animal Care. rats can develop a preference for flavoured solutions associated with recovery from illness [19,20], several lines of reasoning Immunosuppressive Treatment preclude the aforementioned inference on the perception of Mice were assigned into four groups (n = 15 mice/group), therapeutic effect and induction of ‘‘self-medication’’ in the studies according to substrain (MRL/lpr vs. MRL +/+) and treatment above. Namely, impaired taste avoidance in conditioned autoim- (CY vs. vehicle, Veh). For a 12-week period, all mice had ad lib mune MRL/lpr mice [16] could be confounded by deficits that access to a top-mounted 10 ml plastic syringe containing CY have been subsequently reported in this substrain including altered (‘‘Procytox’’, Baxter, Mississauga, ON) diluted to 0.4 mg/ml in gustatory input [21], increased perseverance [22], and/or altered 16% sucrose solution, or Veh (16% aqueous solution of sucrose). emotional reactivity and motivation [23]. As in the original taste The use of 16% sucrose as the vehicle was based on previous aversion experiments, the study documenting ‘‘voluntary’’ con- evidence that MRL substrains do not differ in sucrose intake in a sumption of CY-laced chocolate milk included 23-hr fluid 48-hr, one-bottle test [24]. The CY/sucrose solution was available deprivation [17], which introduces confounding effects of physi- over 3 days each week. Intake of water, sucrose and CY-laced ological and psychological stress induced by prolonged thirst. sucrose solutions was recorded daily, when syringes were re-filled Moreover, repeated weekly bleedings from the retro-orbital sinus with fresh solution. and anesthesia could also affect recovery and ingestive behaviour in the two groups. In the third study, where mice were fluid non- Behavioural Battery deprived, mean volume of CY-laced chocolate milk consumed on Behavioural data were collected between the 21st–22nd weeks of weekdays was merely ,1 ml higher in a large cohort of diseased age, or before overt manifestations of SLE-like disease (e.g., MRL/lpr mice (N = 86) when compared to age-matched controls, alopecia, dermatitis, necrotic ears) develop in the MRL/lpr group N = 78 [18]. Although intake was compared between two [11]. Anxiety-like response to an elevated space was assessed in the substrains and genders, no comparisons were made between mice step-down test as the latency to descend from a raised platform exposed to CY and vehicle only, or shown as a preference over onto a black cloth in a brightly-lit room [23]. Blunted responsive- plain water intake, which was available during weekends only. In ness to sucrose solutions in a choice-paradigm is proposed to summary, such an invasive, single-choice, cross-sectional approach reflect impaired sensitivity to palatable stimulation, and as such, to precludes a clear answer on whether increased CY consumption in model ‘‘anhedonia’’ [25–27]. A brief sucrose preference test was diseased mice is due to differences in thirst levels, responsiveness to given on the days when water was available only, as described a palatable solution/protein content, or indeed – to an altered previously [24]. To examine the functional status of the immune status. If the therapeutic effect of CY is perceived and an hippocampus [28], spontaneous T-maze alternation was assessed adaptive behavioural strategy is learned, one may expect increased using the discrete-trial procedure [29]. Muscle strength and preference for a therapeutic drug as the autoimmune disease sensorimotor coordination were evaluated using a Rota-Rod progresses. Using a continuous choice paradigm and ad lib access (ENV-575M, Med Associates Inc., St. Albans, VT), which to tap water, we presently examine this possibility by longitudinally accelerated from 4 to 40 RPM over the course of 5 min. The assessing the preference for CY-laced sucrose solution in non- latency to fall in three daily trials was recorded. Increased deprived, age-matched MRL/lpr and MRL +/+ mice. A more immobility (floating) in the forced swim test was measured with appropriate design may employ aqueous CY solution, thus EthoVision XT 8 software package (Noldus, NL) and defined as a eliminating the caloric and incentive properties of sucrose. ,5% change in surface area between successive samples. In However, due to its unpalatable, ‘‘metallic’’ taste and negligible addition to the above behavioural battery, spontaneous behaviour intake of CY dissolved in water [18], we mixed CY with 16% in custom-made activity cages equipped with running wheels was sucrose solution to avoid protein content, increase palatability and monitored between 3:00–5:00 P.M. Live-tracking of animal thus achieve the therapeutic dose range of CY (,70–100 mg/ locomotion was performed by EthoVision XT 8. After each b.w.). Functional effects of voluntary CY and sucrose intake were session, mice were returned to their home-cage and activity boxes assessed using a battery of behavioural tests and immunopatho- were cleaned with Quatricide disinfectant (1:256 dilution, PRL logical measures. Pharmacal, Naugatuck, CT).

Methods Immunopathology At 22 weeks of age, mice were anaesthetized with a ketamine/ Animals xylazine mixture, exsanguinated by severing the inferior vena Thirty 8 week old MRL/lpr (stock 485) and 30 age-matched cava, and intracardially perfused with phosphate buffered saline. MRL/MpJ (MRL +/+, stock 486) male mice were purchased from Brains were extracted and sera were stored at 220uC until further the Jackson Laboratories (Bar Harbour, ME). Upon arrival, they analysis. Extracted organs were weighed on an analytical balance were tail-tattooed for identification purposes (AIMS Inc., Hornell, (Mettler Toledo AB54-S; VWR Scientific of Canada, Mississauga, NY) and housed in groups of 5 mice/cage under standard ON). Wet spleen mass was recorded to assess splenomegaly laboratory conditions (light period from 7:00 A.M. –7:00 P.M., (enlarged spleen), a reliable indicator of autoimmune disease in food and water ad libitum). Starting at 11 weeks of age, mice were lupus-prone mice [15]. In addition, serum levels of anti-dsDNA, individually housed 3 days per week for assessment of ingestive anti-cardiolipin and anti-proteinase 3 (PR3) autoantibodies were behaviour and body weight. Two MRL +/+ mice were excluded quantified using a fully automated ELISA analyzer (EUROIM- from the study due to excessive inter-male aggression. Loss .20% MUN Analyzer I). Briefly, 100 ml of each sample (serum 1:50 of body mass, necrotic ear tips, and severe alopecia/dermatitis dilution in sample buffer) was transferred into the corresponding were exclusion criteria and signs for early euthanasia. Three microtiterplate well (EUROIMMUN pre-coated microtiterplate). MRL/lpr mice were sacrificed due to disease manifestations, thus For anti-dsDNA analysis, each well contained antigen substrate of reducing the sample size to N = 55. All procedures were approved dsDNA complexed with nucleosomes and coupled to the solid

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phase. Quantification of anti-cardiolipin antibodies was performed (Substrain6Treatment: F1,51 = 13.666, p,.001; Figure 2A). using wells coated with purified cardiolipin isolated from bovine When sucrose solutions were available, the presence of CY heart. For assessment anti-PR3 antibodies, microplate wells were significantly reduced overall intake, particularly in the MRL/lpr th coated with a mixture of recombinant and native PR3. Irrelevant group after the 11 week of age (Treatment: F1,51 = 672.76, p, of the antibody tested, each sample was incubated for 30 min at .001; Substrain6Treatment6Age: F1,51 = 3.253, p = .031; room temperature and then washed three times with 450 mlof Figure 2B). Consequently, the weekly CY dose consumed via working strength wash buffer. One hundred microliters of 1:2000 voluntary drinking was higher in less symptomatic MRL +/+ than diluted rabbit anti-mouse IgG-HRP conjugate (Promega) was in MRL/lpr mice with early disease manifestations (100–150 mg/ pipetted into each of the microtiterplate wells, left to incubate, and kg in +/+ mice vs. 60–100 mg/kg b.w. in lpr mice; Substrain: washed to remove unbound HRP enzyme conjugate. Subsequent- F1,27 = 28.322, p, .001; Figure 3). Similar results were obtained ly, 100 ml of 3, 3, 5, 5 tetramethylbenzidine enzyme/substrate when group differences in fluid consumption were offset by solution was pipetted into each well of the microtiterplate and calculating preference as intake of sweetened solutions/total fluid incubated for another 20 min at room temperature. One hundred intake. Although preference for CY/sucrose was higher in the CY microliters of stop solution was added to each well in the same lpr group than in the CY +/+ group at 11 weeks of age (U = 40, order and at the same speed as when the chromogen/substrate p,.005), it declined in the subsequent week and remained the solution was introduced. The microtiterplate was shaken at a lowest of all groups throughout the study (Substrain6Treatmen- speed of 20 Hz for 5 s to ensure a homogeneous distribution of the t6Age: F11,561 = 3.553, p,.001; Figure 4). Contrary to our solution. Optical density was determined at a wavelength of expectation, their preference for CY was far below that of the 450 nm and a reference wavelength of 620 nm within 10 min of diseased Veh lpr group, making these two groups even more adding the stop solution. Observed results are expressed as relative dissimilar than the MRL +/+ groups (Substrain6Treatment: optical densities. F1,51 = 24.614, p,.001). Other behaviours. CY treatment significantly reduced step- Statistical Analysis down latency of MRL/lpr mice, but had the opposite effect on Normal distribution of the data was tested by the Shapiro-Wilk congenic controls (Substrain6Treatment: F1,51 = 5.582, p,.05, test. In cases when data departed from normality, the overall Figure 5A). Body weight at 21 weeks correlated positively with assumption was that parametric tests (such as ANOVA and total sucrose intake in the brief preference test (r55 = 0.685, p, ANOVA with repeated measures) were robust enough to detect .001). Expectedly, a significant effect of body weight on significant group differences because the cohorts were indepen- performance was observed with ANCOVA (Weight: dent, sample sizes were equal, and population variances were F1,50 = 8.094, p,.01). Nevertheless, sucrose intake in autoimmune comparable, as revealed by Levine’s test. Analysis of variance MRL/lpr mice was generally lower than in control MRL +/+ (ANOVA) and Analysis of Covariance (ANCOVA) were used in mice (Substrain: F1,50 = 4.327, p,.05). Moreover, there was a overall analysis, with Substrain (MRL/lpr vs. MRL +/+) and trend for CY-treated mice to ingest less of the solution in Treatment (CY vs. Veh) as between-group factors. Age, Concen- comparison to the Veh groups (Treatment: F1,50 = 3.792, p = .057; tration or Trials were considered within-group factors in ANOVA Figure 5B). with repeated measures. Student’s t-test was used in post-hoc Rota-Rod data revealed that CY exposure improved perfor- comparisons. Considering a significant reduction in body weight mance of MRL/lpr mice, but hindered that of MRL +/+ mice was noted when habituation and the behavioural battery was (Substrain6Treatment: F1,51 = 5.379, p,.05; data not shown). A applied (20–22 weeks of age), body weight was considered a significant negative correlation between body weight and time covariate in ANCOVA when a significant correlation between spent on the accelerating rod suggested that smaller mice behavioural performance and weight was observed. In instances performed better (Trial 1: r52 = 20.394, p,.01; Trail 2: r52 = 2 involving non-homogeneity of variance or departure from normal 0.540, p,.001; Trial 3: r52 = 20.401, p,.01). Indeed, repeated distribution, nonparametric tests (e.g., Kruskal-Wallis, Mann- measures ANCOVA with body weight as a covariate revealed a Whitney) were used. Pearson’s correlation was used to examine significant effect of body weight on the latency to fall (Weight: linear relationships between behavioural measures, body weight, F1,50 = 9.370, p,.01), thus accounting for the above group and immunological parameters. Statistical significance was set at difference (Substrain6Treatment: F1,51 = 3.409, n.s.). p#.05 for all comparisons. Graphs indicate mean values and 6 With the present sample size, chronic CY treatment failed to SEM with significant differences of p#.05, p,.01 and p,.001, normalize increased immobility in MRL/lpr mice (Substrain: shown as *, **, and ***, respectively. All calculations were F1,50 = 5.419, p = .024, Figure 5C). Moreover, no significant performed using SPSS 20 software package (SPSS Inc., Chicago, treatment effect was noted in other variables including distance IL). swam, velocity, and overall swimming topography (data not shown). In spontaneous T-maze alternation, all groups performed Results comparably in both acquisition and reversal trials (data not In general, MRL/lpr males were lighter than controls shown), suggesting CY does not affect hippocampal-dependent spatial memory and learning in MRL substrains. Conversely, throughout the study (Substrain: F1,51 = 25.461, p,.001), but prolonged exposure to CY further reduced their body weight at an prolonged exposure to CY prevented the decline in spontaneous ambulatory activity in MRL/lpr mice, but had a detrimental effect older age (Substrain6Treatment6Age: F11,561 = 4.245, p,.001; Figure 1). on the control MRL +/+ group (Substrain6Treatment: 3.1. Ingestive behaviour. Total daily fluid intake ranged F1,42 = 22.661, p,.001; Figure 5D). While CY was unable to from ,7–11 ml in young mice and declined to ,7–9 ml when abolish substrain difference in running wheel activity (Substrain: F = 27.113, p,.001; Figure 5E), it did improve the perfor- they got older (Age: F1,51 = 9.232, p,.001). Although MRL/lpr 1,42 mice exposed to CY drank comparable volumes of water as the mance of treated MRL/lpr mice in comparison to the Veh lpr conspecifics exposed to sucrose, the CY +/+ control group group (t22 = 22.255, p = .034). displayed significantly lower intake than the Veh +/+ group

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Figure 1. Mean body weight from 11–22 weeks of age. Autoimmune MRL/lpr mice were significantly lighter when compared to congenic controls. However, prolonged immunosuppression further reduced their body weight, but not in MRL +/+ mice. doi:10.1371/journal.pone.0100684.g001

Immunopathology dose of CY ingested (a measure independent of the preference) was Chronic immunosuppressive treatment prevented splenomegaly lower in diseased MRL/lpr mice than in CY +/+ group. in MRL/lpr mice (Substrain6Treatment: F1,52 = 11.350, p,.001, Therefore, the notion that autoimmune mice consume solutions Figure 6A). Normalized spleen mass was accompanied by that have been paired or laced with CY in an ‘‘effort to correct significantly lower serum anti-dsDNA (Substrain6Treatment: their immune system dysregulation’’ [18] could not be confirmed F1,55 = 20.933, p,.001, Figure 6B), anti-cardiolipin (Treatment: with the present set of results. As such, this study is in line with a F1,52 = 29.001, p,.001, Figure 6C), and anti-PR3 autoantibody contrarian view on ‘‘dietary wisdom’’ in that was levels in this group (K3 = 37.141, p,.001, U = 8.0, P,0.001 largely ‘‘attributed both to overreliance on theory and to lack of Figure 6D). In summary, unrestricted exposure to sweetened CY critical attention to data’’ [32]. solution abolished severe signs of systemic autoimmunity. How- If a therapeutic effect of an immunosuppressant can be ever, it did not prevent well-documented low brain weight in the perceived and learned indeed, one may expect that diseased MRL/lpr substrain (data not shown) [30,31]. MRL/lpr mice would drink CY dissolved in water. However, this was not observed by Grota and colleagues, prompting the authors Discussion to dilute CY in chocolate milk to improve its palatability [18]. In the current study, we counteracted the taste of CY with 16% The current study tested the hypothesis that zoopharmacognosy sucrose solution and observed comparable CY consumption in the is operational in laboratory mice affected by a progressive, chronic first week of exposure (Figure 3). This confirmed that taste disease. The overall premise was that when autoimmune/ sensitivity to CY was comparable in 11 week-old MRL/lpr mice inflammatory disease manifests, MRL/lpr mice will increase and age-matched MRL +/+ controls. Subsequent decrease in preference for an immunosuppressive drug because of learned intake/preference of the CY lpr group however, does not seem to therapeutic consequences. Compared to studies which involved reflect nutritional or learning deficit. This conclusion is based on classical conditioning and fluid-deprivation [17,18], we used a evidence that diseased MRL/lpr mice show preference for rather ‘‘ethological approach’’ by providing ad lib access to water carbohydrate-rich food [e.g., Veh lpr group performance, and immunosuppressive CY solution. Such a design enabled Figure 4; [33]] and do not display generalized learning deficit experimental animals to freely choose between the solutions, akin [16,34]. Therefore, one may assume that reduced preference for to the unrestricted access to diverse fluids and foods in natural CY-laced sucrose mixture and impaired performance in the brief habitats available to wild mice. However, our results do not sucrose preference test in both drug-treated groups are attributable support self-medication in laboratory mice, because none of the to the repelling and cytotoxic properties of CY. Moreover, it is three expectations were met. Firstly, MRL/lpr mice did not documented that even acute exposure to CY reduces the number increase their consumption of CY-laced sucrose solution alongside of taste buds and impairs responsiveness to sucrose in mice disease progression. Secondly, they failed to display higher [35,36]. In the MRL/lpr strain however, severe systemic disease preference for CY-laced sucrose solution in comparison to per se may further lead to reduced gustatory nerve responses [21] diseased Veh lpr and asymptomatic CY +/+ controls. Lastly, the

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Figure 2. Daily fluid consumption from 11–22 weeks of age. (A) Despite fluctuations in daily water intake over the course of the study, Veh MRL +/+ mice consumed the most water in the 2-bottle test. They also drank significantly more than their CY +/+ counterparts, a trend that was not noted in the MRL/lpr substrain. (B) Lacing sweetened solution with CY significantly reduced daily consumption. However, autoimmune MRL/lpr mice consumed significantly less CY solution in comparison to MRL +/+ mice, particularly after the first week of treatment. doi:10.1371/journal.pone.0100684.g002

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Figure 3. Weekly CY dose ingested. Although both substrains received similar CY dose at the starting of the treatment period, voluntary drinking in subsequent weeks was higher in less symptomatic MRL +/+ than in MRL/lpr mice. doi:10.1371/journal.pone.0100684.g003 and depressive-like behaviour [23,24], which may jointly aggra- The current findings are also in line with the study testing the vate their response to sucrose in the present study. notion of ‘‘dietary wisdom’’ in diabetic rodents [37]. Specifically, laboratory rats affected by a diabetes-like disease failed to develop

Figure 4. Weekly preference for sweet solutions. Initially, CY lpr mice displayed a higher preference for CY/sucrose in comparison to control CY +/+ group at 11 weeks of age. However, this difference was transient and became the lowest of all groups by the end of the study. doi:10.1371/journal.pone.0100684.g004

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Figure 5. Behavioural effects of chronic exposure to CY/sucrose solution. (A) Chronic immunosuppression reduced step-down latency in diseased MRL/lpr mice, but had the opposite effect on congenic controls. (B) In addition to the previously-documented substrain differences in sucrose consumption [24], CY had a significant detrimental effect on the performance in both groups. (C) Although a trend was noted, CY could not abolish previously-reported differences in overall floating in the forced swim test [23]. (D) Prolonged immunosuppression improved spontaneous ambulatory activity in MRL/lpr mice, but had a detrimental effect on MRL +/+ controls. (E) Conversely, CY exposure significantly improved (but did not normalize) running wheel activity in MRL/lpr mice. doi:10.1371/journal.pone.0100684.g005 a strong preference for high fat diets, proposed to have beneficial autoimmune phenotype (http://jaxmice.jax.org/strain/006825. post-ingestional properties. In the MRL strain, one may hypoth- html) and severity of behavioral deficits in the MRL/ lpr substrain esize that altered genetic make-up after repeated inbreeding [40,41]. impaired the adaptive survival mechanisms operational in wild Although zoopharmacognosy could not be shown in the present animals. In support of this hypothesis, inbreeding has been shown study, two other important concepts emerged from the data to have significant detrimental effects on the survivorship of mice collected. The first is the causal link between autoimmunity and reintroduced into a natural habitat [38], offspring survival and aberrant behaviour. CY proved to be a potent immunosuppressant reproductive success [39]. Indeed, the effects of laboratory that abolished systemic immunopathology, as shown by reduced inbreeding can be exemplified by the gradual decline in the spleen weight and autoantibody levels in MRL/lpr mice. These

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Figure 6. Immunosuppressive effects of continuous exposure to CY/sucrose solution. Generalized immunosuppression in CY-exposed MRL/lpr mice evidenced by normalized spleen weight (A), as well as significant reductions in serum levels of autoantibodies directed towards dsDNA (B), cardiolipin (C), and PR3 (D) antigens. doi:10.1371/journal.pone.0100684.g006 findings are consistent with previous observations that brief, 1-hr The second concept that emerged relates to the non-invasive access to CY-laced chocolate milk over 3–4 weeks results in administration route of a noxious drug in experimental mice. significant reductions in lymphadenopathy and serum anti-ssDNA Indeed, the present results suggest that lacing it with a palatable antibody levels [17,18], as well as with generalized immunosup- ingredient can produce a therapeutic dose comparable to the pression after repeated 100 mg/kg/week intraperitoneal CY injection route. As shown in Figure 3, the weekly dose voluntarily injections [42]. Along the same line, ad lib access to the ingested was relatively constant in each substrain and can likely be immunosuppressive solution abolished several behavioural deficits, adjusted by increasing the number of exposure days and/or as shown previously for anxiety-like and motivated behaviours concentration of sucrose or the drug itself. By avoiding repeated following CY treatment [24,42]. Conversely, the lack of effective- exposure to handling, oral gavage, and injections, such an ness in preventing excessive floating in the forced swim test and approach can minimize the confounding effects of stress and brain atrophy suggest a role for non-immunological factors, such reduce variance, thus increasing consistency and precision across as genetic lesions and/or neuroendocrine changes [43]. behavioural and physiological studies.

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Author Contributions reagents/materials/analysis tools: DM BS. Contributed to the writing of the manuscript: MK BS. Conceived and designed the experiments: MK BS. Performed the experiments: MK HZ RH MM. Analyzed the data: MK BS. Contributed

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PLOS ONE | www.plosone.org 9 June 2014 | Volume 9 | Issue 6 | e100684